IDENTIFICATION OF 2 FUNCTIONALLY DISTINCT LYSINE-BINDING SITES IN KRINGLE-37 AND IN KRINGLES 32-36 OF HUMAN APOLIPOPROTEIN(A)

The well documented association between high plasma levels of lipoprotein(a) (Lp(a)) and cardiovascular disease might be mediated by the lysine binding of apolipoprotein(a) (apo(a)), the plasminogen-like, multikringle glycoprotein in Lp(a), We employed a mutational analysis to localize the lysine-binding domains within human apo(a), Recombinant apo(a) (r-apo(a)) with 17 plasminogen kringle IV-like domains, one plasminogen kringle V-like domain, and a protease domain or mutants thereof were expressed in the human hepatocarcinoma cell line HepG2. The lysine binding of plasma Lp(a) and r-apo(a) in the culture supernatants of transfected HepG2 cells was analyzed by lysine-Sepharose affinity chromatography. Wild type recombinant Lp(a) (r-Lp(a)) revealed lysine binding in the range observed for human plasma Lp(a). A single accessible lysine binding site in Lp(a) is indicated by a complete loss of lysine binding observed for r-Lp(a) species that contain either a truncated r-apo(a) lacking kringle IV-37, kringle V, and the protease or a point-mutated r-apo(a) with a Trp 4174 --> Arg substitution in the putative lysine-binding pocket of kringle IV-37, Evidence is also presented for additional lysine-binding sites within kringles 32-36 of apo(a) that are masked in Lp(a) as indicated by an increased lysine binding for the point mutant (Cys-4057 --> Ser), which is unable to assemble into particles, An important role of these lysine-binding site(s) for Lp(a) assembly is suggested by a decreased assembly efficiency for deletion mutants lacking either kringle 32 or kringles 32-35.